In the recent years, development of nanometer sized structures has received much attention for various molecular biological applications. Gold is probably the most suited element because it exhibits a high chemical stability (noble metal), is characterized by its ability to strongly absorb the visible light at definite wavelengths and is intrinsically not toxic. The thiol (R—SH) modified oligonucleotides serve as attractive tools with a vast number of potential applications in the field of nucleic acid chemistry such as it enables covalent attachment of variety of ligands that contain a) α,β-unsaturated ketone; b) maleimide c) other Michael acceptor groups or d) cysteines in proteins to make disulfide bonds. In addition to this, thiol has a strong specific interaction with gold surface to form reversible covalent bond with gold.
The reactive thiol group can be introduced into oligonucleotides by incorporating sulfide modified phosphoramidite monomers during oligonucleotide synthesis. Generally, two different types of sulfide modified monomers viz disulfide stratagy [Jones, D. S., Hachmann, J. P., Conrad, M. J., Coutts, S., Livingston, D. A. U.S. Pat. No. 5,391,785, 1995] or S-trityl protection [Connolly, B. A.; Rider, P. Nucleic Acids Res. 1985 13, 4485] are very popular to achieve this. Reactive thiol group from the disulfide is generated by treating oligo with reducing agent such as dithiothreitol (DTT). Whereas, in the other S-trityl strategy, it is generated by cleaving trityl group by silver nitrate. However, this strategy has clear disadvantage of elaborate cleavage process of trityl group with the silver nitrate, which results in relatively poor yields of the final oligonucleotide. Hence, disulfide modified phosphoramidites serve as superior probes for generating thiol groups. The most popular disulfide probes are with the general formula DMT-O—R—S—S—R—O—P(CE)(NiPr2), where R being C3 or C6 (3, chart 1) spacer arm [Jones, D. S., Hachmann, J. P., Conrad, M. J., Coutts, S., Livingston, D. A. U.S. Pat. No. 5,391,785, 1995]. The synthesis of phosphoramidites 3 (where n=6, Chart 1) and supports 4 (where n=6, Chart 1) with C6 arm is simple and high yielding [Jones, D. S., Hachmann, J. P., Conrad, M. J., Coutts, S., Livingston, D. A. U.S. Pat. No. 5,391,785, 1995]. However, synthesis of C3 disulfide phosphoramidite compound 3 (Chart 1, where n=3) has recently been reported very briefly by Yosuke Taniguchia et. al. [Taniguchi, Nitta, A., Park, S. M., Kohara, A., Uzu, T., Sasaki, S. Bioorg. Med. Chem. 2010, 18, 8614]
The reported synthetic protocol was not reproducible in generating the target compound 3 (Chart 1, where n=3) in our hands and purification procedure was not reported. We therefore carried out detailed investigation to develop a new synthetic and purification method that gives phosphoramidite 3 (Chart 1, where n=3) in a high purity for commercial, research and development. Our optimized synthetic protocol is reproducible, suitable for multi gram scale and yields target phosphoramidite 3 (Chart 1, where n=3) in high purity by 31P NMR (>94%). [Srivastava, S. C.; Thatikonda, S. K.; Srivastav, S. K. Shukla, P. U.S. Patent Application No. 2012/000103, 2012]

Nuzzo and Allara have discovered that reactive thiol group adsorb on gold surface and forms ordered mono layers. [Nuzzo, R. G., Allara, D. L. Jour. Am. Chem. Soc. 1983, 105, 4481]. After this, oligonucleotides with thiol group are very much used to generate self assembled monolayers (SAMs) on the gold surfaces. Although different molecules can be immobilized (silanes, carboxylic acids, pyridines, sulphites and thiols) on different surfaces (gold, silver, platinum, copper, mercury and glass), chemisorption of thiols on gold is a common and simple procedure to immobilize probes on a surface. DNA functionalized gold nanoparticles have since become widely used building blocks in key nucleic acid based assembly strategies and serve as unique probes for recognizing specific sequences in DNA segments [Storhoff, J. J., Elghanian, R., Mucic, R. C., Mirkin, C. A., and Letsinger, R. L. J. Am. Chem. Soc. 1998 120, 1959] as a building blocks for assembling novel structures and materials [Mucic, R. C., Storhoff, J. J., Mirkin, C. A., Letsinger, R. L. J. Am. Chem. Soc. 1998 120, 12674] and bio diagnostics and nano technology based therapeutics [Merkins, C. A., Letsinger, R. L., Mucic, R. C., Storhoff, J. J. Nature, 1996, 382, 607; Hurst, S. J., Hill, H. D., Mirkin, C. A. J. Am. Chem. Soc. 2008, 130, 12192]. It has been proven that formation of these monolayers is influenced by several factors such as temperature, solvent, buffer concentration, chain length of the adsorbate, cleanliness of the substrate, rate of reaction with the surface and the reversibility of adsorption of the components of the monolayer. These applications depend on the reversible association of gold and sulfur bond between the attached oligonucleotide and nano particle.
The oligonucleotides attached with single thiol group are unstable during the washing steps and formation of stable attachment of oligonucleotides is very important property for its success in applications such as for DNA chip technology. The covalent bond between gold and sulfur is in the order of magnitude from 30 to 40 Kcal/mol, which is relatively weak in order to anchor a biopolymer onto a surface. [Dubois L. H., Zegarski B. R., Nuzzo R. G. Proc. Natl. Acad. Sci. USA 1987 84 4739; Liepold, P., Kratzmüller, T., Persike, N., Bandilla, M., Hinz, M., Wieder, H., Hillebrandt, H., Ferrer, E., Hartwich, G. Anal Bioanal Chem, 2008, 391, 1759-1772]. It's been reported that oligonucleotides that are conjugated with mono functional thiol group are slowly lost at higher temperatures and also in the presence of high salt concentration buffers [Li, Z., Jin, R., Mirkin, C. A., Letsinger, R. L. Nucleic Acids Res. 2002, 30, 1558]. The stability studies by Letsinger et. al. on SAMs of oligonucleotides that are conjugated to gold surface by mono thiol group revealed that these are completely displaced by treating with the buffers containing DTT [Letsinger, R. L., Elghanian, R., Viswanadham, G., Mirkin, C. A. Bioconj. Chem. 2000, 11, 289]. This feature limits applications of these probes in solutions containing thiols such as a PCR solution that has DTT as a stabilizer for the polymerase enzyme.
So there is strong need to develop novel disulfide compounds that are capable of forming stable SAMs of oligonucleotides for wider biological applications. One can anticipate that stability of mono layers could be increased by multiple numbers of gold-sulfur bonds per oligonucleotides. There have been few reports [Letsinger, R. L., Elghanian, R., Viswanadham, G., Mirkin, C. A. Bioconj. Chem. 2000, 11, 289-291; Hartwich, G., Frischmann, P., Ferrer, E., U.S. Pat. No. 7,601,848, 2002; Seliger, H., Prokein, T. U.S. Patent No 2005/0059728, 2004] that has introduced novel thiol modifiers which can generate multiple thiol groups per oligonucleotide. Its been proved that SAMs produced by these modifications are much more stable than corresponding SAMs generated by mono thiol modifier in buffers containing DTT [Letsinger, R. L., Elghanian, R., Viswanadham, G., Mirkin, C. A. Bioconj. Chem. 2000, 11, 289].
The task of the present invention is to make novel disulfide monomer that can introduce poly functional thiol groups for immobilization or labeling. Herein, we describe five membered disulfide (dithiolane) based anchoring group 1 (Chart 1) for the introduction of one two thiol groups. This dithiolane modification is simple to synthesize, is broadly useful, and can potentially afford gold-oligonucleotide conjugates that exhibit greater stability. Previously, similar dithiolane based phosphoramidites and supports have been reported [Seliger, H., Prokein, T. U.S. Patent No 2005/0059728, 2004] for the synthesis of dithiolane probes. However, even though they claimed with longer spacer arm, reported synthesis was with shorter spacer arm. The successful attachment of oligonucleotides onto gold surface ideally requires relatively longer spacer arm. The other important issue with the previously reported dithiolane phosphoramidites is that, spacer arm is asymmetric branching chain with a secondary hydroxyl group carrying DMT group for oligo chain synthesis. Close proximity of hydroxyl group to the phosphodiester bond might result in nucleophilic attack, which further leads to chain cleavage of oligonucleotide strand. Similar side reaction leading to nucleophilic attack and subsequent chain cleavage of synthetic RNA is also very likely. Thus the quality of the dithiolane oligonucleotides and probes can be compromised. If multiple dithiolane moieties are attached into a defined sequence DNA or RNA, multiple scissions of oligonucleotides is possible and serious compromise of DNA or RNA is possible. To address these issues, we have planned a new probe design that has longer spacer arm.
We have designed a symmetrical linker such that free hydroxyl group will be generated at one carbon away from the phosphodiester group at 3′ or 5′ end of oligonucleotides. Symmetrical branching type of linker, as it placed away from the phosphodiester, avoids chain cleavage possibilities. With these improvements, we strongly believe that our probes are designed to be ideally suited to application in high quality probes consisting of dithiolane moieties.